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The Journal of Neuroscience, October 15, 2000, 20(20):7855-7860
Cholinergic Inhibition of Ventral Midbrain Dopamine Neurons
Christopher D.
Fiorillo and
John T.
Williams
Vollum Institute, L474, Oregon Health Sciences University,
Portland, Oregon 97201
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ABSTRACT |
Muscarinic acetylcholine receptors are common throughout the
CNS. The predominant subtypes in the brain are positively
coupled to phosphoinositide hydrolysis and have been found to modulate multiple conductances. Muscarinic receptor activation is most often
observed to be excitatory because of suppression of various potassium
conductances. Here it is reported that three distinct effects of
muscarinic receptor activation can be observed in isolation from one
another, depending on the duration of receptor activation and the
concentration of agonist. Brief activation of muscarinic receptors, as
is likely to occur with normal synaptic transmission, hyperpolarized
dopamine neurons of the ventral midbrain through a calcium-activated
potassium conductance. With repeated or persistent activation of
muscarinic receptors, the hyperpolarizing response was entirely
desensitized in the absence of any change in resting membrane
potential. With sustained activation by higher concentrations of
agonist, dopamine neurons were depolarized. This demonstrates that
muscarinic receptors can mediate very diverse, and even opposing, postsynaptic effects on neurons depending on the pattern of
acetylcholine release.
Key words:
IPSP; calcium-activated potassium (sK); phosphoinositide; calcium stores; heterologous desensitization; ryanodine
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INTRODUCTION |
Five subtypes of muscarinic
acetylcholine receptors have been cloned to date, all of which fall
into one of two categories of G-protein-coupled receptor (for review,
see Caulfield, 1993 ). The M2-like receptor family (m2, m4) inhibit
adenylyl cyclase and Ca2+ channels and
activate inwardly rectifying K+ channels.
M1-type receptors (m1, m3, and m5) are the more common type of
muscarinic receptor in the CNS. These receptors are positively coupled
to phospholipase C, which catalyzes the production of inositol
trisphosphate (IP3) and diacylglycerol, which can
then trigger release of calcium from intracellular stores and
activation of protein kinase C, respectively. M1 type receptors have
been found to modulate a surprisingly large number of conductances, even in a single neuron. In CA1 pyramidal neurons of the hippocampus, muscarinic receptors have been shown to block at least four potassium conductances (Cole and Nicoll, 1984; Nakajima et al., 1986; Madison et
al., 1987), to activate at least two potassium conductances (Segal
1982; Wakamori et al., 1993; Zhang et al., 1992), and to activate a cation conductance (Colino and Halliwell, 1993). The transduction mechanisms mediating most of these effects are poorly understood. Likewise, it is not known to what extent these conductances are modulated simultaneously or whether they are modulated selectively under distinct conditions. It has been shown that the concentration of
muscarinic agonist can determine which of three potassium conductances are blocked (Madison et al., 1987). In addition, muscarinic receptors may produce activation before inhibition of potassium conductance or
conductances (Segal 1982; Wakamori et al., 1993). It is not known which
factors determine whether excitation or inhibition predominates. We
have investigated these issues in dopamine neurons of the ventral midbrain.
Previous work has shown that stimulation of muscarinic receptors
activates dopamine neurons (Scarnati et al., 1986; Lacey et al., 1990;
Gronier and Rasmussen, 1998) (for review, see Kitai et al., 1999).
Superfusion of muscarine in a ventral midbrain slice activates M1-type
receptors to produce depolarization of dopamine neurons, most likely
through inhibition of a potassium conductance and an increase in a
cation conductance (Lacey et al., 1990). Like the M1-type of muscarinic
receptor, metabotropic glutamate receptors 1 and 5 (mGluR1 and mGluR5)
couple to phosphoinositide (PI) hydrolysis and mobilization of calcium
from intracellular stores. It was recently shown in dopamine neurons of
the ventral midbrain that mGluR1 mediates a slow IPSP through
mobilization of calcium from intracellular stores (Fiorillo and
Williams, 1998). The hyperpolarizing action of mGluRs was readily
desensitized by slow application of agonist, lost with whole-cell
recording techniques using high calcium-buffering solutions, and
blocked by a nonspecific action of the GABAA
receptor antagonist bicuculline. For all of these reasons, an analogous
hyperpolarizing action of muscarinic receptors could have gone
undetected in previous studies. The present study demonstrates that
rapid application of acetylcholine activates muscarinic receptors on
dopamine neurons to release calcium from internal stores, which
subsequently increases an apamin-sensitive potassium conductance.
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MATERIALS AND METHODS |
Slice preparation. Intracellular recordings were made
in horizontal slices (250- to 300-µm-thick) of ventral midbrain from adult male Wistar rats (150-200 gm). Details of the method of slice
preparation and recording have been published (Williams et al., 1984).
Recordings were made from submerged slices in a chamber (0.5 ml)
superfused with physiological saline at a rate of 1.5 ml/min and
maintained at 35°C. The solution was equilibrated with 95%
O2 and 5% CO2 and
contained (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 2.4 CaCl2, 1.4 NaH2PO4, 25 NaHCO3, and 11 D-glucose.
Recordings. Intracellular recordings were made with an
Axoclamp 2A amplifier (Axon Instruments, Foster City, CA) and
were used for studies involving cholinergic IPSPs and hyperpolarizing responses to pressure-applied acetylcholine. Microelectrodes (50-70 M ) were filled with 2 M KCl. During
experiments, the membrane potential was adjusted to between  60 and
70 mV to prevent spontaneous action potentials. Dopamine neurons of
the ventral tegmental area (VTA) and substantia nigra pars compacta
(SNc) were identified by their electrical properties (Johnson and
North, 1992). Dopamine neurons fired spontaneous, relatively
long-duration action potentials at a rate of 1-5 Hz, each followed by
a large afterhyperpolarization. Dopamine neurons also displayed a
time-dependent depolarization in response to hyperpolarizing current
injection. No differences were observed in responses of neurons in the
SNc and the VTA; the data were pooled. Whole-cell recordings were made
with an Axopatch 1D and were used for studies involving mGluR IPSCs and iontophoresis of acetylcholine. Patch pipettes contained (in
mM): 115 K-methylsulfate, 20 NaCl, 1.5 MgCl2, 5 HEPES, 0.1 EGTA, 2 ATP, 0.5 GTP, and 10 phosphocreatine. Electrode resistance was 2-5 M; access
resistance ranged between 7 and 15 M and was compensated by 80%. The somata of individual cells were visualized with an upright
microscope and infrared illumination. Dopamine neurons were identified
both visually and by the presence of large H-currents at hyperpolarized
potentials. The current in response to iontophoretic application of
acetylcholine and synaptically released transmitter was measured at a
holding potential of 50 mV.
Synaptic responses. Synaptic potentials or currents were
evoked with bipolar tungsten stimulating electrodes with a tip
separation of 300-600 µm. A train of 8-10 stimuli of 400 µsec at
0.3-1.5 mA was delivered at 66 Hz (15 msec interval) every 60 sec.
Stimulating electrodes were placed within 1 mm caudal of the recording
site. A cocktail of antagonists was present in experiments on synaptic potentials. This included picrotoxin (100 µM;
GABAA), strychnine (1 µM; glycine),
NBQX (5 µM; AMPA), MK-801 (50-100 µM
pretreatment only; NMDA), eticlopride (100 nM; D2), and CGP
56999a (100-300 nM; GABAB) or CGP
35348 (200 µM; GABAB).
Drugs. Drugs were applied to the slice by superfusion,
except acetylcholine, which was applied by pressure ejection (in
experiments with intracellular recording) or by iontophoresis (in
experiments with whole-cell recording). For pressure ejection,
acetylcholine (3-30 mM) in 0.09% NaCl solution was
applied from patch pipettes (1.5 µm tip diameter) for 3-300 msec at
20 psi using a Picospritzer II. Pipettes were moved closer to the cell
body until a hyperpolarizing response was elicited. The duration of the
ejections was usually adjusted to maximize the hyperpolarizing response
while minimizing the depolarizing response; this helped to prevent
desensitization of the hyperpolarizing response. In all experiments
with pressure ejection, hexamethonium (200 µM) was
present to block nicotinic acetylcholine receptors. For iontophoresis,
acetylcholine (1 M) was applied (50-100 nA, 100 msec) from
a microelectrode placed within 10 µm of the cell body. Hexamethonium
was not used; the nicotinic response did not interfere with the slower
muscarinic response and was used as a measure of acetylcholine release
from the iontophoretic pipette.
Apamin, (±)muscarine chloride, picrotoxin, (±)scopolamine,
strychnine, and tetrodotoxin were from Sigma (St. Louis, MO).
Acetylcholine chloride, caffeine, S()-eticlopride,
hexamethonium, and MK-801 were from Research Biochemicals International
(Natick, MA).
1,2,3,4-tetrahydro-6-nitro-2,3,-dioxo-benzo[f]quinoxaline (NBQX) and
(S)-3,5-dihydroxyphenylglycine (DHPG) were from
Tocris Cookson (St. Louis, MO). Ryanodine was from Calbiochem (San
Diego, CA). CGP 56999a and CGP 35348 were a gift from Novartis
Pharmaceuticals (Basel, Switzerland).
Data analysis. Values are given as arithmetic means ± SEM. The percent change produced by a superfused drug was
calculated as the mean amplitude of three to five responses after
equilibrium had been reached (5-10 min) relative to the mean of five
responses before drug superfusion. Paired comparisons were made using a Wilcoxon signed-rank test.; p < 0.05 was considered as
a significant difference. Unless stated otherwise, all drug effects
quantified are statistically significant.
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RESULTS |
Synaptic potentials
Stimulating electrodes were positioned caudal to the dopamine cell
region, in an attempt to stimulate ascending cholinergic fibers. In the
presence of ionotropic receptor antagonists and a
GABAB receptor antagonist, a train of 5-10
stimuli at 66 Hz elicited slow IPSPs, as previously reported (Fiorillo
and Williams, 1998). In 15 of 16 randomly selected neurons, superfusion
of the slice with the muscarinic receptor antagonist scopolamine (1 µM) had little or no effect on the IPSP. In one cell,
scopolamine blocked the IPSP. This IPSP appeared distinct from the
scopolamine-insensitive IPSPs (mediated by mGluRs) in that it had a
relatively short latency to onset. In subsequent experiments,
scopolamine was tested only on IPSPs that had a similar, short-onset
latency. In all three such cases (of ~50 cells), scopolamine was
effective in blocking the IPSP. An example of a scopolamine-sensitive
IPSP is shown in Figure 1. Scopolamine
blocked the early component of the IPSP and left a slower
mGluR-mediated IPSP. The amplitude of the four scopolamine-sensitive
IPSPs was 10.0 ± 2.5 mV, and the latency to peak was 307 ± 39 msec. In contrast, the latency to peak of the mGluR IPSP averaged
~500 msec and was never <300 msec (Fiorillo and Williams, 1998,
their Fig. 1D). Although under the
experimental conditions tested scopolamine was rarely effective, it
appears that it is possible to evoke cholinergic IPSPs mediated by
muscarinic receptors in at least some dopamine neurons. By contrast,
slow EPSPs were rarely observed in dopamine neurons with intracellular recording, and when present they are blocked by mGluR antagonists, but
not muscarinic receptor antagonists (Shen and Johnson, 1997; Fiorillo
and Williams, 1998).
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Figure 1.
A slow IPSP blocked by scopolamine. This figure is
an intracellular recording of membrane potential. A train of electrical
stimuli were applied at the arrow to evoke transmitter
release. Traces 1-3 are each the average of three
trials. 1, The slow IPSP in the presence of ionotropic
receptor antagonists (glutamate and GABA). 2, Addition
of the GABAB receptor antagonist CGP 35348 (200 µM) blocked only a small fraction of the IPSP in this
cell. 3, Addition of the muscarinic receptor antagonist
scopolamine (1 µM) blocks an early component of the IPSP,
leaving a later component that was mediated by glutamate acting on
mGluRs. 4, The scopolamine-sensitive component of the
IPSP, isolated by subtraction of trace 3 from trace
2.
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Hyperpolarization to exogenous acetylcholine
To further investigate the actions of muscarinic receptors on
dopamine neurons, acetylcholine (3-30 mM) was applied
briefly (3-300 msec) from patch pipettes by pressure ejection, in the presence of the nicotinic receptor antagonist hexamethonium (200 µM). Acetylcholine had both hyperpolarizing and
depolarizing actions (Fig.
2A). Short applications
resulted in hyperpolarization only (Fig. 2A), whereas
longer applications resulted in a hyperpolarization followed by a
longer-lasting depolarization (Fig. 2A). The
mechanism of the hyperpolarization was further investigated
pharmacologically.
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Figure 2.
Multiple actions of acetylcholine on dopamine
cells. A, This is an intracellular recording of membrane
potential. Pressure ejection of acetylcholine for 10 msec evoked
primarily a hyperpolarization, whereas a 50 msec pulse evoked an
equivalent hyperpolarization, followed by a longer-lasting
depolarization. The depolarization was insensitive to hexamethonium,
but was blocked by scopolamine (data not shown). Each trace represents
the average of two or three trials. The depolarization in response to
50 msec acetylcholine evoked an action potential on its rising phase,
which is diminished in amplitude because of averaging.
B, This is a whole-cell recording of current obtained in
voltage clamp. Iontophoretic application of acetylcholine evoked an
early short-latency inward current (nicotinic) followed by a slower
outward current (muscarinic). The amplitude of the nicotinic component
increased as the membrane potential was held more negative, whereas the
outward current decreased in amplitude. C, A summary of
experiments obtained in voltage clamp as shown in B. The
amplitude of the nicotinic and muscarinic currents are plotted as a
function potential.
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By analogy with previous work on mGluRs in dopamine neurons, we tested
apamin, a potent and selective blocker of a subset of
small-conductance, calcium-activated potassium channels (SK2 and SK3;
Kohler et al., 1996). Apamin (100 nM) effectively
blocked the hyperpolarization produced by acetylcholine (86 ± 18%; n = 4). Voltage-clamp experiments using the
whole-cell configuration were used to further characterize the
conductance that was activated by acetylcholine. By using an internal
solution with minimal calcium buffering capacity, a reproducible
hyperpolarizing response to acetylcholine was observed. Acetylcholine
applied by iontophoresis induced a short-latency inward current
followed by a slower outward current (Fig. 2B). The
short-latency inward current was blocked by hexamethonium (data not
shown), indicating that it resulted from the activation of nicotinic
receptors. The slower outward current was blocked by scopolamine (data
not shown). The outward muscarinic current declined in amplitude as the
holding potential was shifted to more negative potentials, suggesting
that it resulted from the activation of a potassium conductance. The
amplitude of nicotinic current increased with hyperpolarization (Fig.
2B,C). Taken together the results suggest that
acetylcholine acts on a muscarinic receptor to activate an
apamin-sensitive potassium conductance that mediates a
hyperpolarization of the membrane potential.
Like the mGluR1 receptors, M1-type muscarinic receptors expressed on
dopamine cells (Lacey et al., 1990) are known to induce calcium release
from intracellular stores. The effect of caffeine was used to further
characterize the hyperpolarization induced by acetylcholine. Caffeine
increases calcium release from intracellular stores, apparently by
greatly increasing the calcium sensitivity of the ryanodine receptor
(Ehrlich et al., 1994). Superfusion of caffeine (1 mM)
reversibly enhanced the amplitude of acetylcholine hyperpolarizations
(90 ± 47%; n = 4; Fig.
3). At a higher concentration, caffeine
(10 mM) reversibly inhibited the acetylcholine
hyperpolarization (73 ± 9%; n = 6; Fig. 3),
presumably by depleting ryanodine-sensitive calcium stores or by
blocking the IP3 receptor directly. Ryanodine (10 µM), which locks the ryanodine receptor in a
subconductance state, depletes calcium stores (Bezprozvanny et al.,
1991) and blocked the acetylcholine-induced hyperpolarization measured
with intracellular recordings (80 ± 7%; n = 4)
and the outward current measured with whole-cell recordings (65 ± 11%; n = 6). When ruthenium red (30 µM), a blocker of the ryanodine receptor, was
included in the whole-cell pipette solution, the outward muscarinic
current was not blocked, but the effect of ryanodine was significantly reduced from 65 ± 11% (n = 6) to 21 ± 6% (n = 4; p < 0.05). The observation
that acetylcholine still induced an outward current in the presence of
ruthenium red suggests that ryanodine receptors were not required for
this muscarinic response. Taken together, the results suggest that
muscarinic receptors increase intracellular free calcium by release
from a store that is sensitive to depletion by ryanodine but that does
not require the activation of ryanodine receptors. Similar results were
obtained with ruthenium red in experiments where photolysis of
caged-IP3 evoked an increase in intracellular
calcium (Morikawa et al., 2000). The conclusion was that the activation
of IP3 receptors released calcium from a store
that was sensitive to depletion by ryanodine.
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Figure 3.
Activation of muscarinic receptors induces calcium
release from intracellular stores. Caffeine facilitates and blocks the
acetylcholine-induced hyperpolarization, depending on the
concentration. These experiments were obtained with intracellular
recording of membrane potential. On the left is a plot
showing the time course of the facilitation and inhibition of the
acetylcholine hyperpolarization induced by caffeine. Each point
represents the average amplitude of responses from several cells, each
of which was normalized to the average amplitude of the five responses
preceding application of caffeine. The control acetylcholine responses
were 7.3 ± 0.9 (n = 4) and 10.7 ± 1.9 (n = 5) in experiments with 1 and 10 mM
caffeine, respectively. On the right are raw traces
illustrating the modulation by caffeine of acetylcholine
hyperpolarizations. Each trace is the average of two to four trials.
Top, Facilitation by 1 mM caffeine.
Bottom, Inhibition by 10 mM caffeine.
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Desensitization and depolarization by sustained activation of
muscarinic acetylcholine receptors and metabotropic glutamate
receptors
Previous studies, in dopamine and other neurons, have reported
depolarization caused by activation of PI-coupled muscarinic receptors.
The depolarization was often accompanied by a decrease in potassium
conductance. Typically these studies examined responses to sustained
activation of muscarinic receptors, and it is possible that under such
conditions a calcium-mediated inhibitory response was desensitized. To
examine this possibility, voltage-clamp experiments were used to
determine the effect of a low concentration of muscarine (300 nM) on the hyperpolarization and outward current induced by
rapidly applied acetylcholine. In experiments using intracellular recordings of membrane potential, the hyperpolarizing responses to
pressure-applied acetylcholine were reduced by 74 ± 3%
(n = 4; Fig.
4A) without
significantly altering resting membrane potential (+1.4 ± 0.7 mV). This concentration of muscarine is approximately tenfold lower
than the half-maximal concentration of muscarine effective in
depolarizing the membrane (Lacey et al., 1990). In voltage-clamp
experiments with whole-cell recordings, muscarine (300 nM) caused a small inward current (26 ± 3 pA; n = 10) and reduced the outward current induced by
acetylcholine by 76 ± 4% (n = 10; Fig.
4B).
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Figure 4.
The acetylcholine response is potently blocked by
activation of PI-coupled receptors. A, An intracellular
recording of the hyperpolarization induced by pressure ejection of
acetylcholine in the absence (control) and presence of muscarine (300 nM). The plot below is a summary of several similar
experiments. Superfusion of a low concentration of muscarine (300 nM) or the mGluR1 agonist DHPG (1 µM) almost
entirely blocked hyperpolarizations evoked by pressure-applied
acetylcholine. Wash of either agonist rapidly produced partial
recovery; the remaining desensitization did not recover after 15 min of
wash. The control acetylcholine responses were 6.2 ± 0.4 mV
(n = 4) and 6.9 ± 1.7 mV
(n = 5) in experiments with muscarine and DHPG,
respectively. B, A voltage-clamp recording of outward
current induced by a synaptically activated mGluR IPSC followed
by the outward current induced by iontophoretically applied
acetylcholine. The mGluR IPSC and the outward current induced by
acetylcholine were dramatically and reversibly reduced during the
superfusion of muscarine (300 nM). The nicotinic response
was not affected.
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Because mGluR1 and muscarinic receptors found on dopamine neurons are
thought to couple to similar effectors, the effect of superfusion of a
low concentration of an mGluR agonist on the muscarinic-induced
hyperpolarization was examined. Superfusion of the mGluR1 agonist DHPG
(1 µM) depolarized the membrane by 3.4 ± 0.8 mV and
reduced the acetylcholine hyperpolarization by 73 ± 11%
(n = 5; Fig. 4B). After washing DHPG
or muscarine for 10-15 min, the acetylcholine response partially
recovered to a new steady-state. Partial recovery was also seen from
desensitization of mGluR1-mediated hyperpolarizations by DHPG (Fiorillo
and Williams, 1998). Thus, the hyperpolarizing response induced by
muscarinic receptor activation was desensitized by a heterologous mechanism.
The effect of muscarine on the outward current induced by mGluRs was
used to further examine heterologous desensitization. The mGluR IPSC
was reduced by 72 ± 9% (Fig. 4B;
n = 5) by superfusion with muscarine (300 nM). Thus, it appears that continuos activation of either muscarinic receptors or mGluRs with low agonist
concentrations results in a potent and reversible heterologous
desensitization. The kinetics of heterologous desensitization was
examined using the mGluR IPSC and the outward current induced by
iontophoretically applied acetylcholine. When acetylcholine was applied
4 sec before a mGluR IPSC was evoked, the amplitude of the IPSC was
reduced to 78 ± 6% (n = 4) of control (Fig.
5). When the protocol was reversed, such
that the IPSC was evoked 4 sec before the application of acetylcholine,
the outward current in response to acetylcholine was reduced to 81 ± 4% (n = 4) of control. Thus, even a brief activation of either receptor can result in desensitization that outlasts the acute response.
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Figure 5.
Transient receptor activation causes heterologous
desensitization that outlasts the acute response. These are current
recordings obtained in the whole-cell configuration.
Top, Two superimposed traces showing an example of an
experiment in which iontophoretic application of acetylcholine either
did nor did not precede the activation of a mGluR IPSC.
Bottom, Two superimposed traces in which the protocol
was reversed such that an mGluR IPSC preceded the iontophoretic
application of acetylcholine. The currents at the beginning of each
trace are the response to a step hyperpolarization of 10 mV (200 msec).
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DISCUSSION |
The present results demonstrate that brief activation of
muscarinic receptors activated a potassium conductance to hyperpolarize the membrane potential of ventral midbrain dopamine neurons. The outward current was mediated by mobilization of calcium from
intracellular stores and activation of an apamin-sensitive potassium
conductance. With longer activation of muscarinic receptors, the
muscarine-induced outward current desensitized, and an inward current
that mediated depolarization was produced. A previous report of the
effect of muscarinic agonists on dopamine neurons in vitro
found only depolarization (Lacey et al., 1990). This was apparently
attributable to the relatively slow and sustained application of
agonists, as was found in the present study with superfusion.
Muscarinic receptors on dopamine neurons therefore modulate multiple
channels to cause either inhibition or excitation, in a manner similar
to mGluR1 (Fiorillo and Williams, 1998).
Evidence was presented that the muscarinic inhibition could be
activated by synaptically released acetylcholine. Although muscarinic
IPSPs were observed only in a relatively small fraction of cells, these
IPSPs were fairly large (5-15 mV). The reason for the rarity of
cholinergic IPSPs is unknown, but may be consistent with cholinergic
innervation of a minority of dopamine neurons (Garzon et al., 1999). On
the other hand, the increase in potassium conductance in response to
exogenously applied acetylcholine was observed in almost every dopamine
cell. Thus, it may be that the horizontal sections of the ventral
midbrain are not well suited for the activation of afferents from the
cholinergic fibers arising from the pedunculopontine nucleus. No
muscarinic EPSPs were observed in the present or previous studies (Shen
and Johnson, 1997), which is likely attributable to the longer duration
of receptor activation necessary to cause depolarization. At most, only
very small nicotinic receptor-mediated EPSPs are present in dopamine
neurons (J. Dani, personal communication). In spite of the
results obtained in brain slices, experiments done in vivo
have found that injection of atropine, a muscarinic antagonist, into
the VTA increased dopamine release in the nucleus accumbens (E. Munn
and R. Wise, personal communication). This could be caused by
blockade of cholinergic IPSPs in dopamine neurons.
Like mGluR1, muscarinic receptors produce hyperpolarizing or
depolarizing responses in dopamine neurons, depending on the duration
of time for which they are active (Fig. 2A).
Desensitization of muscarinic receptor-mediated hyperpolarization
occurs after a transient hyperpolarizing response and is almost
complete with sustained activation of the receptor. This heterologous
desensitization can occur in the absence of any changes in resting
membrane potential. Although agonist-induced activation of potassium
conductance is almost entirely blocked, activation of the
apamin-sensitive afterhyperpolarization (AHP) in dopamine neurons is
unaltered by these same agonists (Lacey et al., 1990; Fiorillo and
Williams, 1998). Because the apamin-sensitive AHP in dopamine neurons
is partially dependent on ryanodine-sensitive calcium stores
(our unpublished observations), and assuming a single population
of apamin-sensitive channels, it would appear that the desensitization
occurs upstream of the ryanodine-sensitive stores. Desensitization
could occur through depletion of IP3-sensitive
calcium stores (Irving et al., 1992), which are known to be easily
depleted in many cell types, and can recover quickly with calcium
influx (Berridge, 1998).
The actions of muscarinic receptors (and mGluRs) in dopamine neurons
appear similar to their actions in CA1 hippocampal pyramidal neurons.
In CA1, muscarinic receptors mediate both hyperpolarizing and
depolarizing responses (Segal 1982; Wakamori et al., 1993), and the
hyperpolarizing response is dependent on intracellular calcium stores
(Wakamori et al., 1993). Studies using slow application of agonist
found only excitatory responses (Madison et al., 1987). Desensitization
of the hyperpolarizing response has not been examined in the CA1.
However, activation of both muscarinic receptors and mGluRs is known to
block the slow AHP in these cells (Cole and Nicoll, 1984;
Madison et al., 1987; Desai and Conn, 1991). Like the muscarinic
hyperpolarization observed in the present study, the slow AHP in CA1 is
calcium-dependent, and at least in some types of neurons involves the
release of calcium from intracellular stores (Sah and McLachlan, 1991).
In CA1 neurons, these calcium-dependent processes are blocked by
concentrations of muscarinic agonists that are substantially below
those necessary to depolarize the membrane (Cole and Nicoll, 1984;
Madison et al., 1987). Thus, it appears to be the case in a number of
neuronal types that brief activation of muscarinic receptors
hyperpolarized neurons through release of calcium, whereas tonic
activation by even very low levels of agonist desensitize this response
and in some cells decreased the slow AHP. Stronger receptor stimulation
always caused depolarization. Although possible, there is no evidence
that these distinct postsynaptic actions of muscarinic receptors (or
mGluRs), described in dopamine neurons or CA1 pyramidal neurons, result from activation of distinct receptors.
Cholinergic neurotransmission in the CNS may occur primarily in a
diffuse, nonsynaptic fashion, rather than classical point-to-point synaptic transmission (Descarries et al., 1997). It has been
hypothesized that, at least in regions of dense cholinergic
innervation, an ambient level of acetylcholine is maintained, resulting
in tonic activation of receptors (Descarries et al., 1997). It is shown here that tonic activation of muscarinic receptors by low
concentrations of agonist blocks the calcium-mediated activation of
potassium currents by both muscarinic receptors and mGluRs. To observe
inhibition by acetylcholine, the ambient concentration would have to be
less than ~30 nM, or else the inhibitory response would
be desensitized. This estimate is based on the concentration of
acetylcholine that is equipotent to 300 nM muscarine
(concentration used in the present study) in activating a
calcium-dependent potassium conductance in dissociated CA1 pyramidal
neurons (Wakamori et al., 1993). Quantitative microdialysis performed
in the striatum found 18 nM acetylcholine in the
extracellular space (Vinson and Justice, 1997), consistent with levels
found in other brain regions (Descarries et al., 1997). Thus, it is
suggested that the calcium-dependent potassium conductance is
dynamically regulated by acetylcholine, being phasically activated
during periods of relatively low cholinergic activity and tonically
blocked during periods of sustained cholinergic input. A second
potentially important functional consequence of the cholinergic
innervation of dopamine cells could be in the modulation of
mGluR-mediated inhibition through heterologous desensitization.
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FOOTNOTES |
Received June 27, 2000; revised Aug. 3, 2000; accepted Aug. 9, 2000.
This work was supported by National Institutes of Health Grants DA
04523 and DA 05793. We thank Drs. Carlos Paladini and Hitoshi Morikawa
for helpful comments on this manuscript.
Correspondence should be addressed to Dr. John T. Williams, Vollum
Institute, L474, Oregon Health Sciences University, 3181 Southwest Sam
Jackson Park Road, Portland, OR 97201. E-mail: williamj{at}ohsu.edu.
Dr. Fiorillo's present address: Institute for Physiology, University
of Fribourg, Rue de Musèe 5, CH 1700 Fribourg, Switzerland.
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REFERENCES |
-
Berridge MJ
(1998)
Neuronal calcium signaling.
Neuron
21:13-26[Web of Science][Medline].
-
Bezprozvanny I,
Watras J,
Ehrlich B
(1991)
Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum.
Nature
351:751-754[Medline].
-
Caulfield MP
(1993)
Muscarinic receptors-characterization, coupling and function.
Pharmacol Ther
58:319-379[Web of Science][Medline].
-
Cole AE,
Nicoll RA
(1984)
Characterization of a slow cholinergic postsynaptic potential recorded in vitro from rat hippocampal pyramidal neurons.
J Physiol (Lond)
352:173-188[Abstract/Free Full Text].
-
Colino A,
Halliwell JV
(1993)
Carbachol potentiates Q current and activates a calcium-dependent non-specific conductance in rat hippocampus in vitro.
Eur J Neurosci
5:1198-1209[Web of Science][Medline].
-
Desai MA,
Conn PJ
(1991)
Excitatory effects of ACPD receptor activation in the hippocampus are mediated by direct effects on pyramidal cells and blockade of synaptic inhibition.
J Neurophysiol
66:40-52[Abstract/Free Full Text].
-
Descarries L,
Gisiger V,
Steriade M
(1997)
Diffuse transmission by acetylcholine in the CNS.
Prog Neurobiol
53:603-625[Web of Science][Medline].
-
Ehrlich BE,
Kaftlan E,
Bezprozvanny S,
Bezprozvanny I
(1994)
The pharmacology of intracellular Ca2+-release channels.
Trends Pharmacol Sci
15:145-149[Medline].
-
Fiorillo CD,
Williams JT
(1998)
Glutamate mediates an inhibitory postsynaptic potential in dopamine neurons.
Nature
394:78-82[Medline].
-
Garzon M,
Vaughan RA,
Uhl GR,
Kuhar MJ,
Pickel VM
(1999)
Cholinergic axon terminals in the ventral tegmental area target a subpopulation of neurons expressing low levels of the dopamine transporter.
J Comp Neurol
410:197-210[Web of Science][Medline].
-
Gronier B,
Rasmussen K
(1998)
Activation of midbrain presumed dopaminergic neurones by muscarinic cholinergic receptors: an in vivo electrophysiological study in the rat.
Br J Pharmacol
124:455-464[Web of Science][Medline].
-
Irving AJ,
Collingridge GL,
Schofield JG
(1992)
L-glutamate and acetylcholine mobilise Ca2+ from the same intracellular pool in cerebellar granule cells using transduction mechanisms with different Ca2+ sensitivities.
Cell Calcium
13:293-301[Web of Science][Medline].
-
Johnson SW,
North RA
(1992)
Two types of neurone in the rat ventral tegmental area and their synaptic inputs.
J Physiol (Lond)
450:455-468[Abstract/Free Full Text].
-
Kitai ST,
Shepard PD,
Callaway JC,
Scroggs R
(1999)
Afferent modulation of dopamine neuron firing patterns.
Curr Opin Neurobiol
9:690-697[Web of Science][Medline].
-
Kohler M,
Hirschberg B,
Bond CT,
Kinzie JM,
Marrion NV,
Maylie J,
Adelman J
(1996)
Small-conductance, calcium-activated potassium channels from mammalian brain.
Science
273:1709-1714[Abstract/Free Full Text].
-
Lacey MG,
Calabresi P,
North RA
(1990)
Muscarine depolarizes rat substantia nigra zona compacta and ventral tegmental neurons in vitro through M1-like receptors.
J Pharmacol Exp Ther
253:395-400[Abstract/Free Full Text].
-
Madison DV,
Lancaster B,
Nicoll RA
(1987)
Voltage clamp analysis of cholinergic action in the hippocampus.
J Neurosci
7:733-741[Abstract].
-
Morikawa H,
Imani F,
Khodakhah K,
Williams JT
(2000)
Inositol 1,4,5-triphosphate-evoked responses in midbrain dopamine neurons.
J Neurosci
20:RC103 (1-5).
-
Nakajima Y,
Nakajima S,
Leonard RJ,
Yamaguchi K
(1986)
Acetylcholine raises excitability by inhibiting the fast transient potassium current in cultured hippocampal neurons.
Proc Natl Acad Sci USA
83:3022-3026[Abstract/Free Full Text].
-
Sah P,
McLachlan EM
(1991)
Ca2+-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca2+-activated Ca2+ release.
Neuron
7:257-264[Web of Science][Medline].
-
Scarnati E,
Proia A,
Compama E,
Pacitti C
(1986)
A microiontophoretic study on the nature of the putative synaptic neurotransmitter involved in the pedunculopontine-substantia nigra pars compacta excitatory pathway of the rat.
Exp Brain Res
62:470-478[Web of Science][Medline].
-
Segal M
(1982)
Multiple actions of acetylcholine at a muscarinic receptor studied in the rat hippocampal slice.
Brain Res
246:77-87[Web of Science][Medline].
-
Shen KZ,
Johnson SW
(1997)
A slow excitatory postsynaptic current mediated by G-protein-coupled metabotropic glutamate receptors in rat ventral tegmental dopamine neurons.
Eur J Neurosci
9:48-54[Web of Science][Medline].
-
Vinson PN,
Justice JB
(1997)
Effect of neostigmine on concentration and extraction fraction of acetylcholine using quantitative microdialysis.
Neurosci Methods
73:61-67[Web of Science][Medline].
-
Wakamori M,
Hidaka H,
Akaike N
(1993)
Hyperpolarizing muscarinic responses of freshly dissociated rat hippocampal CA1 neurones.
J Physiol (Lond)
463:585-604[Abstract/Free Full Text].
-
Williams JT,
North RA,
Shefner S,
Nishi S,
Egan TM
(1984)
Membrane properties of rat locus coeruleus neurones.
Neuroscience
13:137-156[Web of Science][Medline].
-
Zhang L,
Weiner J,
Carlen PL
(1992)
Muscarinic potentiation of IK in CA1 hippocampal neurons: electrophysiological characterization of the transduction pathway.
J Neurosci
12:4510-4520[Abstract].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20207855-06$05.00/0
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ABSTRACT
INTRODUCTION
